Synthetic oligodeoxynucleotides have been shown to be sequence inhibitors of gene expression, and thus may be useful in the experimental therapy of malignant disease. However, very little is known about the cellular pharmacology of these agents. Such knowledge is vital because in order for these agents ever to become clinically important, their efficacy must be optimized. Little information is currently available on 1) kinetic and equilibrium parameters of cell surface binding; 2) rates and mechanisms of internalization; 3) intracellular distribution; and 4) rates and mechanisms of oligo efflux. We hypothesize that HL60 cells, despite some encouraging literature results, manifest de novo "resistance" to the effects of oligonucleotides. We hypothesize that cellular defense mechanisms include 1) oligo-induced diminution in PKC kinase activity, which promotes exocytosis; 2) compartmentalization of oligos in a non- acidic environment and lack of penetration of oligo into deep cellular compartments (e.g., the lysosome) which leads to; 3) rapid efflux of full length material so that the concentration of intracytoplasmic oligo is, at most, only a few percent of the external concentration. We will test our hypotheses in HL60 cells: We will use a standard, phosphodiester 15-mer homopolymer of thymidine which has been 5' labeled with fluorescein, to treat the cells. We will then monitor rates of endocytosis and exocytosis using flow cytometry. By this method, we will determine the kinetics of efflux, determine the number of efflux compartments, and calculate the kinetic constants A, alpha,B,beta etc. We will determine the intracellular concentration of oligomer by synthesizing a double labeled (fluorescein + 32p) oligonucleotide. We will perform similar experiments with uncharged, 3H-labeled methylphosphonate oligomers. We will increase intracellular oligo retention by using dequalinium and/or probenecid. We will correlate changes in efflux kinetics with changes in antisense efficacy. This will be done by using an oligo complementary to the c-myc mRNA, and by examining levels of c-myc protein by immunoprecipitation and gel electrophoresis.